A kind of expression method and application of soluble recombinant insulin and its derivatives

An expression method and insulin technology, which is applied to the expression method and application field of soluble recombinant insulin and its derivatives, can solve the problems affecting the cost of insulin and its derivatives, poor insulin solubility, cumbersome purification steps, etc., and achieve soluble expression level And the progress of production technology, the effect of increasing the yield of soluble expression and improving the yield of expression

Active Publication Date: 2015-11-18
NANJING UNIV
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AI Technical Summary

Problems solved by technology

Although insulin and its derivatives have been put into production, the problems of cumbersome purification steps and low yield have always been the key problems affecting the cost of insulin and its derivatives
Some scientists have tried to express human insulin and thioredoxin (Trx) fusion (Zhang Shoutao et al., Biotechnology, 2010, 20(2):14-16; Dong Wenfu et al., Herbivorous Livestock, 2006, 4:4-6 ), but the experimental results show that the solubility of insulin expressed by fusion of thioredoxin (Trx) is still poor

Method used

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  • A kind of expression method and application of soluble recombinant insulin and its derivatives
  • A kind of expression method and application of soluble recombinant insulin and its derivatives
  • A kind of expression method and application of soluble recombinant insulin and its derivatives

Examples

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Embodiment

[0025] 1. Construction of insulin glargine expression vector

[0026] The plasmids cloned with the A chain (pUC57-A) and B chain (pUC57-B) of insulin glargine were synthesized and constructed by GenScript. Enterokinase cleavage sites were added to the N-terminal of insulin glargine A chain and the C-terminal of B chain, respectively.

[0027] The Trx gene was amplified from the plasmid pET32a by PCR. The upstream primer was: 5'-CTTGCGGGATCCATGAGCGATAAAATTATTCACC-3', and the downstream primer was: 5'-GTTATAATACCCAAGCTTGGCCAGGTTAGCG-3'. The recognition sequences of restriction endonucleases BamHI and HindIII were introduced into the 5' ends of the upstream and downstream primers, respectively. The PCR product was recovered by 1% agarose gel electrophoresis, and then double digested with BamHI and HindIII, and ligated with the vector pUC57-B that had undergone the same double digestion. The pB-Trx plasmid was obtained, the pB-Trx plasmid was digested with EcoRI and HindIII, and...

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Abstract

The invention belongs to the biotechnological field and in particular relates to soluble recombinant insulin, an expression method of an insulin derivative and an application of the insulin. The expression method is used for effectively improving gene engineering expression yield of the soluble recombinant insulin and the insulin derivative. When the expression method is applied with low-temperature expression and multi-molecule couple co-expression, the yield of the soluble recombinant insulin and the derivative thereof can be remarkably improved. The invention further provides a purifying preparation method of the soluble recombinant insulin and the derivative thereof as well as application of the insulin in preparation of a medicine for treating diabetes mellitus.

Description

1. Technical field: [0001] The present invention belongs to the field of biotechnology. Specifically, it relates to a method for expressing soluble recombinant insulin and its derivatives and its application. 2. Background technology: [0002] In modern society, diabetes is a chronic disease with high incidence, many complications and difficult to cure. Worldwide, the incidence of diabetes is on the rise. The disease is a leading cause of blindness, kidney failure, amputations, heart disease and stroke. It kills as many patients each year as it kills HIV / AIDS. At present, my country may have become the country with the largest number of diabetic patients. The total number of adults with diabetes in my country is 92.4 million, including 43.1 million in rural areas and 49.3 million in urban areas. The incidence rate exceeds 10%, and the diabetes drug market has exceeded 10 billion yuan. [0003] Insulin drugs are the best choice for controlling and treating diabetes. Acco...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C07K14/62C07K1/22A61K38/28A61P3/10
Inventor 华子春甘子仪
Owner NANJING UNIV
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